title: A differential proteome screening system for post-translational modification-dependent transcription factor interactions.
abstract: Post-translational modifications (PTMs) of transcription factors alter interactions with co-regulators and epigenetic modifiers. For example, members of the C/EBP transcription factor family are extensively methylated on arginine and lysine residues in short, conserved, modular domains, implying modification-dependent cofactor docking. Here we describe array peptide screening (APS), a systematic and differential approach to detect PTM-dependent interactions in the human proteome using chemically synthesized, biotinylated peptides coupled to fluorophore-labeled streptavidin. Peptides with and without a modified residue are applied in parallel to bacterial expression libraries in an arrayed format. Interactions are detected and quantified by laser scanning to reveal proteins that differentially bind to nonmodified or modified peptides. We have previously used this method to investigate the effect of arginine methylation of C/EBPβ peptides. The method enables determination of PTM-dependent transcription factor interactions, quantification of relative binding affinities and rapid protein classification, all independently of the transactivation potential of peptides or cellular abundance of interactors. The protocol provides a cost-effective alternative to mass spectrometric approaches and takes 3-4 d to complete.
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.
authors.forename: Ole
authors.initials: O
authors.lastname: Pless
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Kowenz-Leutz
authors.forename: Gunnar
authors.initials: G
authors.lastname: Dittmar
authors.forename: Achim
authors.initials: A
authors.lastname: Leutz
mesh_terms: D001120:Arginine / Q000378:metabolism; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism; D020543:Proteome / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D019151:Peptide Library; D010455:Peptides; D011506:Proteins; D020543:Proteome; D014157:Transcription Factors; D001120:Arginine